Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1β, IL-17, and TGF-β production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.


Figure S4 .
Figure S4.Post-Manufacturing CAR T Cell Gating Strategy.(A) Gating strategy for analysis of spectral immunophenotyping flow cytometry data of CAR T cells post-manufacturing.(B) FMO's and representative positive populations depicting positive and negative gates.

Figure S5 .
Figure S5.Expression of CD45RA/CD45RO and CD4/CD8 in MP TRAC-CAR T Cells at Scale.Representative contour plots for expression of (A) CD45RA/CD45RO and (B) CD4/CD8 MP or Control TRAC-CAR T cells (Donor F shown).

Figure S6 .
Figure S6.In Vivo Potency of MP and Control TRAC-CAR T cells.(A) The flux over time (luminescence from IVIS images) is shown for mice treated with MP or Control TRAC-CAR T cells.(B) IVIS images depict tumor growth over time for the same conditions.(2 donors, N MP = 6, N Control = 8).Error bars represent mean and standard deviation.

Figure
Figure S7 Gating Scheme for Analysis of Lymphocytes in Mouse Spleens.Gating strategy for analysis of live, CD5/CD45 + lymphocytes in isolated mouse spleens.(B) FMO's and representative positive populations depicting positive and negative gates.

Figure S8 .
Figure S8.Expression of CD4 and CD8 Post In Vivo Tumor Challenge.(A) Representative contour plots for CD4/CD8 expression in CD5+/CD45+/TCR-/transgene+ lymphocytes isolated from mouse spleens (Green = MP, Donor E; Gray = Control, Donor F; Orange = No CAR Ctrl, Donor E). (B) Bar graphs for relative expression of CD4 and CD8 for MP or Control -CAR T cells and no CAR control T-cells.Samples with less than 20 CD5+/CD45+/TCR-/transgene+ events were excluded from analysis.2 donors, N MP = 10, N Control = 6, N NoCARCtrl = 6.Error bars represent mean and standard deviation.

Figure S10 .
Figure S10.Differentiation of TRAC-CAR T cells in Serial Stimulation Assay.MP and Control TRAC-CAR T cells were serially stimulated with GD2 + CHLA-20 neuroblastoma cells for 20 days, collected, and stained to immunophenotype human T lymphocytes via flow cytometry.(A) Marker expression t-SNE (t-distributed Stochastic Neighbor Embedding) plots of MP and Control TRAC-CAR T cells.These maps were generated via flow cytometry to track CD45RA, CD45RO, CD62L, CCR7, LAG3, and TIGIT expression.Dot plots separate cells by condition, T SCM , or transitional T-cell status.Representative contour plot and bar graphs of double positive populations of (B) CD45RO vs CD45RA and (C) CD62L vs CCR7. 2 donors, N MP = N Control = 4. Error bars represent mean and standard deviation.Statistical significance was determined with paired t-tests; *p<0.05;**p<0.01.

Table S1 . Guide RNA's and Primers Used in Study.
The TRAC gRNAs and forward and reverse primers for amplifying the original GD2-CAR and No CAR Control linear constricts for nanoplasmid construction.(5' --> 3')

Table S3 .
Sanger Sequencing Primers Used in Study.The primers used for performing Sanger sequencing on the GD2-CAR or No CAR Control plasmid are shown.(5' --> 3') (in Excel addendum)

Table S4 .
Antibodies Used in Study for Flow Cytometry.The clone, manufacturer, fluorophore, catalog number, and volume needed per sample are listed for each antibody used for flow cytometry in this study.(in Excel addendum)